Nucleic acids encoding D4 desaturases and D5 elongases

ABSTRACT

Disclosed are methods and compositions related to ONC-T18, D4-desaturases, D5 elongases, their isolation, characterization, production, identification, and use for fatty acid production, as well as organisms containing these compositions and organisms expressing them.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/668,793, filed Jun. 17, 2010, now U.S. Pat. No. 8,765,422,which is a national phase application claiming priority to InternationalPatent Application No. PCT/IB2007/004553, filed Oct. 31, 2007, whichclaims the benefit of U.S. Provisional Patent Application No.60/949,730, filed Jul. 13, 2007, each of which is incorporated herein byreference in its entirety.

I. BACKGROUND

There is overwhelming scientific evidence that (n-3) highly unsaturatedfatty acids such as docosahexaenoic acid (DMA) have a positive effect oncardio-circulatory diseases, chronic inflammations and brain disorders.The (n-6) fatty acids such as eicosapentaenoic acid (EPA) on the otherhand have been noted as intermediate metabolites within the eicosanoidsteroids, such as prostaglandins, leucotrienes or the like.

Currently, the main source of these highly unsaturated fatty acids isfish, with EPA and DHA noted within various blue fish (such as sardinesand tuna) at amounts around 20% and 10%, respectively. It is believedthat such a fatty acid profile occurs through the natural selection ofoptimal ratios for optimal performance within each species of fish. Yet,if one intends to use fish oil as the sole source of these lipids,several disadvantages exist, such as problems with flavor taint,uncontrollable fluctuations in availability and natural fish oil contentvariability. In addition, if one intends to obtain a highly purified(n-3) or (n-6) oil from these sources, it is very difficult topreferentially separate and purify.

Previously disclosed is a Thraustochytriales eukaryote, ONC-T18 andrelated organisms, capable of producing high amounts of DHA and EPA aswell as other preferred fatty acids. ONC-T18 is disclosed inInternational Application PCT/IB2006/003977 and United Statesprovisional applications 60/751,401 and 60/821,084 which are all hereinincorporated by reference for information at least related to ONC-T18and fatty acids produced therein. The manipulation of the DHA and EPApathways is desirable. Disclosed herein is the isolation andcharacterization of two enzymes from ONC-T18 involved in these pathways,a D4 desaturase and a D5 elongase.

II. SUMMARY

Disclosed are methods and compositions related to ONC-T18,D4-desaturases D5 elongases, their isolation, characterization,production, identification, and use for fatty acid production, as wellas organisms containing these compositions and organisms expressingthem.

III. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description illustrate the disclosed compositions and methods.

FIG. 1 shows the pathways of EPA and DHA production.

FIG. 2A shows a phylogentic tree for the isolated D4 desaturase and FIG.2B shows a phlogenetic tree for the isolated D5-elongase.

FIG. 3 shows a genetic outline schematic for the isolated D4 desaturase.

FIG. 4 shows a genetic outline schematic for the isolated D5 elongase.

IV. DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods or specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

A. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

“Primers” are a subset of probes which are capable of supporting sometype of enzymatic manipulation and which can hybridize with a targetnucleic acid such that the enzymatic manipulation can occur. A primercan be made from any combination of nucleotides or nucleotidederivatives or analogs available in the art which do not interfere withthe enzymatic manipulation.

“Probes” are molecules capable of interacting with a target nucleicacid, typically in a sequence specific manner, for example throughhybridization. The hybridization of nucleic acids is well understood inthe art and discussed herein. Typically a probe can be made from anycombination of nucleotides or nucleotide derivatives or analogsavailable in the art.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular D4 desaturase and D5 elongase is disclosed anddiscussed and a number of modifications that can be made to a number ofmolecules including the D4 desaturase and D5 elongase are discussed,specifically contemplated is each and every combination and permutationof D4 desaturase and D5 elongase and the modifications that are possibleunless specifically indicated to the contrary. Thus, if a class ofmolecules A, B, and C are disclosed as well as a class of molecules D,E, and F and an example of a combination molecule, A-D is disclosed,then even if each is not individually recited each is individually andcollectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F,C-D, C-E, and C-F are considered disclosed. Likewise, any subset orcombination of these is also disclosed. Thus, for example, the sub-groupa A-E, B-F, and C-E would be considered disclosed. This concept appliesto all aspects of this application including, but not limited to, stepsin methods of making and using the disclosed compositions. Thus, ifthere are a variety of additional steps that can be performed it isunderstood that each of these additional steps can be performed with anyspecific embodiment or combination of embodiments of the disclosedmethods.

B. Compositions

The use of omega-3 concentrates (eicosapentaenoic acid EPA, 20:5 n-3 anddocosahexaenoic acid DHA, 22:6 n-3) has become important in thefortification of certain foods to promote a healthy diet.Thraustochytrids are marine protists that naturally produce DHA, at upto 20% of their biomass. The capability to ferment these organismsprovides for a renewable, long-term source of these omega-3 oils.Characterisation of the fatty acid metabolic pathway (FIG. 1) revealsthe importance of a D5-elongase responsible for the elongation of EPAinto docosapentaenoic acid (DPA, 22:5 n-3), as well as a D4-desaturasebeing involved in the desaturation of DPA into DHA. Manipulation of thespecific enzyme activity can influence the yield of EPA and DHA producedby our ONC-T18 strain as required by the needs of the market, and ourcustomers.

Using degenerate primers constructed from a conserved region in the genesequence from enzymes of different Thraustochytrid strains(Thraustochytrium sp. (CS020087), Thraustochytrium aureum (AF391546),Thraustochytrium sp. ATCC 34304 (AF391543), Thraustochytrium sp. ATCC21685 (AF489589), and Thraustochytrium sp. FJN-10 (DQ133575) forD4-desaturase; Thraustochytrium sp. (CS160897) and Thraustochytriumaureum (CS160879) for D5-elongase) a D4-desaturase and a D5 elongasefrom ONC T-18 were isolated. Further a portion of the genes from genomicDNA (967 bp and 593 bp, respectively) was PCR amplified. Genome walking(APAgene GOLD kit, BIO S&T, Montreal, Quebec) was used to extend theknown sequence to incorporate the entire open reading frame, and extendfurther to identify the adjacent genes on either side along with thepromoter region. Primers were then constructed for the complete sequenceto produce a full gene PCR product incorporating the entire open readingframe (1758 bp and 1099 bp, respectively). The PCR product was clonedinto pT7-Blue3 vector (Novagen, San Diego, Calif.) and transformed intoEscherichia coli NovaBlue (DE3) (Novagen, San Diego, Calif.) and thensequenced.

Resultant gene plasmids were purified using the UltraClean 6 Minute MiniPlasmid Prep kit (MC) BIO Laboratories, Inc., Solana Beach, Calif.). Thegene inserts were then excised using the restriction enzymes BamHI andNotI and cloned into the pYES2 yeast expression vector (Invitrogen,Carlsbad, Calif.). The new vector constructs identified as pYDes(D4-desaturase) and pYElo (D5-elongase) were then transformed intoSaccharomyces cerevisiae INVSc1 using the S.c. EasyComp Transformationkit (Invitrogen, Carlsbad, Calif.), under the galactose promoter Gal1.The negative control strain was INVSc1 containing the unaltered pYES2vector, and these were grown simultaneously. The vector selection wasdone using the uracil auxotrophy of the yeast strain, SC medium withouturacil was used.

The activity and specificity of the D4-desaturase and D5 elongase weredetermined using the yeast expression system. The transformed yeast wasgrown in SC-U medium containing 2% glucose, 1% Tergitol NP-40 for 48hrs, 150 RPM at 30° C. A substrate medium was prepared containing SC-U,2% galactose, 1% raffinose, 1% Tergitol NP-40 and 500 μM specific freefatty acid. The transformed yeast culture was inoculated into 100 mlsubstrate medium at an OD600 of 0.5 and cultures incubated at 20° C. for5 days. The biomass was recovered by centrifugation at 2000 RPM for 5min, washed once with 100 mM phosphate buffer (pH 7.0), then freezedried. Fatty acid methyl ester gas chromatography was subsequentlycarried out to determine the efficiency of fatty acid desaturation andelongation. The percent conversion of the substrate was determined bycalculating (product)/(substrate+product)*100.

BLASTx results showed the ONC T-18 D4 desaturase to be 96% similar to aThraustochytrium sp. ATCC 21685 D4-desaturase. FIG. 2A shows a rootedneighbour-joining phylogenetic tree, determined using ClustalX,bootstrap analysis (1000×) using the results of a BLASTx search whencompared to the ONC-T18 D4-desaturase sequence. The D4-desaturase genehas an open reading frame of 1560 bp, transcribing a 519 amino acidprotein. Analysis of this protein shows a cytochrome b5 domain withthree histidine box motifs and four transmembrane regions, all elementscharacteristic of front-end desaturase. Adjacent to this gene are fiveputative TATA boxes, multiple repeat regions, two promoters and aprotein kinase identified upstream and an AP2 binding protein downstream(FIG. 3).

Conversely, a BLASTx search identified the D5-elongase protein as having89% identity to a Thraustochytrium sp. FJN-10 polyunsaturated fatty acidelongase. FIG. 2B shows a rooted neighbour-joining phylogenetic tree,determined using ClustalX, bootstrap analysis (1000×) using the resultsof a BLASTx search when compared to the ONC-T18 D5-elongase sequence.Further analysis determined that this 831 bp long elongase, coding a 276amino acid protein, contains four transmembrane regions specific tomitochondria, and one histidine box motif. The upstream component ofthis D5-elongase region comprises a single TATA box, multiple repeatregions and a promoter prior to a mitochondrial import receptor, whiledownstream the beginning of a membrane occupation and recognition nexusmotif was identified (FIG. 4).

Characterisation of pYDes (Table 1) for both the n-3 and n-6 pathways,showed a 14% conversion of DPA n-3 or docosatetraenoic acid (DTA 22:4n-6) to DIM or DPA n-6, respectively.

TABLE 1 D4-desaturase enzyme activity and characterisation % conversionWith 0.01% ferric citrate Substrate Product average stdev (x) %conversion increase DPA DHA 14.04 4.01 4 38.70 2.75 fold DTA DPA n-613.76 1.31 3 33.43 2.43 fold DGLA ARA 0.87 0.27 3

When fed the corresponding D4-desaturase substrate dihomo-g-linolenicacid (DGLA, 20:3 n-6), no conversion was detected. In an effort toincrease pYDes activity, trace metals such as ferric citrate was addedto the media, resulting in an increase in DPA to DHA conversion (Table1). In contrast, presently pYElo shows minimal conversion when eitherEPA or arachidonic acid (ARA 20:4 n-6) were fed.

D4-desaturase and D5-elongase genes from the high fatty acid producingstrain Thraustochytrium sp. ONC-T18, have been successfully isolated andcloned, followed by expression in S. cerevisiae.

Furthermore, the D4-desaturase enzyme was shown to convert both DPA orDTA to their respective end products both in their native form and viasupplementation with trace metals. Feed studies with other fatty acidsconfirmed the D4 specific activity of this desaturase. Through the useof gene manipulation techniques, such as error-prone PCR, this activitywill be further enhanced so as to effect an increase in production ofDHA in our strain.

Disclosed are compositions comprising a D4 desaturase wherein the D4desaturase has at least or greater than 70%, 80%, 89%, 90%, 95%, 96%,97% identity to SEQ ID NO:26.

Also disclosed are compositions, wherein any change away from SEQ IDNO:26 is a conservative change.

Also disclosed are compositions comprising a nucleic acid wherein thenucleic acid encodes any of the D4 desaturase.

Also disclosed are compositions, further comprising a vector.

Also disclosed are compositions comprising a cell wherein the cellcomprises an of the compositions.

Also disclosed are compositions, wherein the cell is a eukaryote, aprokaryote, a Thraustochytrid, a yeast, or an e coli.

Also disclosed are compositions comprising a non-human animal whereinthe non-human animal comprises any of the compositions.

Also disclosed are compositions, wherein the composition produces morepolyunsaturated fatty acids than the composition in the absence of theD4 desaturase.

Also disclosed are compositions, wherein the fatty acid is EPA or DHA.

Also disclosed are compositions, wherein the desaturase contains atleast one histidine box.

Also disclosed are compositions, wherein the desaturase contains atleast 2 histidine boxes.

Also disclosed are compositions, wherein the desaturase contains atleast three histidine boxes.

Also disclosed are compositions, wherein the histidine box comprises thesequence HXXHH (SEQ ID NO:29) where X is any amino acid.

Also disclosed are compositions, wherein the histidine box comprises thesequence QXXHH (SEQ ID NO:30).

Also disclosed are compositions, wherein the desaturase also comprises acytochrome b5 domain.

Also disclosed are compositions, wherein the cytochrome b5 domainresides at the 5′-end.

Also disclosed are compositions, wherein the desaturase is in thepresence of a desaturase substrate

Also disclosed are compositions, wherein the substrate has aconcentration of at least 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600μM, 700 μM, 800 μM, 900 μM, or 1000 μM.

Also disclosed are compositions, wherein the substrate isDocosapentaenoic acid (22:5n-3), Docosatetraenoic acid (22:4n-6), orDihomo-gamma-linolenic acid (20:3n-6).

Also disclosed are compositions, wherein the desaturase converts atleast 0.1%, 0.5%, 1%, 5%, 10%, 30%, 50%, 70%, 90%, 95% of the availablesubstrate.

Also disclosed are compositions, wherein the desaturase converts theamount of substrate shown in Table 1.

Also disclosed are compositions, wherein the composition is isolated.

Also disclosed are compositions comprising a D5 elongase wherein the D5elongase has at least or greater than 70%, 80%, 89%, 90%, 95%, 96%, 97%identity to SEQ ID NO:15.

Also disclosed are compositions, wherein any change away from SEQ ID NO:15 is a conservative change.

Also disclosed are compositions comprising a nucleic acid wherein thenucleic acid encodes any of the D5 elongases.

Also disclosed are compositions encoding elongases or desaturases,further comprising a vector.

Also disclosed are compositions wherein the composition produces morepolyunsaturated fatty acids than the composition in the absence of theD5 elongase, such as DHA or EPA.

Also disclosed are compositions wherein the elongase is in the presenceof an elongase substrate

Also disclosed are compositions, wherein the substrate isEicosapentaenoic acid (20:5n-3) or Arachidonic acid (20:4n-6).

Also disclosed are compositions, wherein the elongase converts at least0.1%, 0.5%, 1%, 5%, 10%, 30%, 50%, 70%, 90%, 95% of the availablesubstrate.

Also disclosed are compositions, wherein the composition is isolated.

Also disclosed are compositions comprising any of the discloseddesaturase compositions and any of the disclosed elongase compositions.

Also disclosed are methods for producing a polyunsaturated fatty acidcomprising using one or more of any of the compositions.

Also disclosed are methods, wherein the fatty acid produced is eitherEPA or DHA.

Also disclosed are methods of producing the compositions comprisingisolating any of the compositions.

1. Sequence Similarities

It is understood that as discussed herein the use of the terms homologyand identity mean the same thing as similarity. Thus, for example, ifthe use of the word homology is used between two non-natural sequencesit is understood that this is not necessarily indicating an evolutionaryrelationship between these two sequences, but rather is looking at thesimilarity or relatedness between their nucleic acid sequences. Many ofthe methods for determining homology between two evolutionarily relatedmolecules are routinely applied to any two or more nucleic acids orproteins for the purpose of measuring sequence similarity regardless ofwhether they are evolutionarily related or not.

In general, it is understood that one way to define any known variantsand derivatives or those that might arise, of the disclosed genes andproteins herein, is through defining the variants and derivatives interms of homology to specific known sequences. This identity ofparticular sequences disclosed herein is also discussed elsewhereherein. In general, variants of genes and proteins herein disclosedtypically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99 percent homology to the stated sequence or the nativesequence. Those of skill in the art readily understand how to determinethe homology of two proteins or nucleic acids, such as genes. Forexample, the homology can be calculated after aligning the two sequencesso that the homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, PASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment. It isunderstood that any of the methods typically can be used and that incertain instances the results of these various methods may differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity, and be disclosed herein.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

2. Hybridization/Selective Hybridization

The term hybridization typically means a sequence driven interactionbetween at least two nucleic acid molecules, such as a primer or a probeand a gene. Sequence driven interaction means an interaction that occursbetween two nucleotides or nucleotide analogs or nucleotide derivativesin a nucleotide specific manner. For example, G interacting with C or Ainteracting with T are sequence driven interactions. Typically sequencedriven interactions occur on the Watson-Crick face or Hoogsteen face ofthe nucleotide. The hybridization of two nucleic acids is affected by anumber of conditions and parameters known to those of skill in the art.For example, the salt concentrations, pH, and temperature of thereaction all affect whether two nucleic acid molecules will hybridize.

Parameters for selective hybridization between two nucleic acidmolecules are well known to those of skill in the art. For example, insome embodiments selective hybridization conditions can be defined asstringent hybridization conditions. For example, stringency ofhybridization is controlled by both temperature and salt concentrationof either or both of the hybridization and washing steps. For example,the conditions of hybridization to achieve selective hybridization mayinvolve hybridization in high ionic strength solution (6×SSC or 6×SSPE)at a temperature that is about 12-25° C. below the Tm (the meltingtemperature at which half of the molecules dissociate from theirhybridization partners) followed by washing at a combination oftemperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the Tm. The temperature andsalt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The conditionscan be used as described above to achieve stringency, or as is known inthe art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is hereinincorporated by reference for material at least related to hybridizationof nucleic acids). A preferable stringent hybridization condition for aDNA:DNA hybridization can be at about 68° C. (in aqueous solution) in6×SSC or 6×SSPE followed by washing at 68° C. Stringency ofhybridization and washing, if desired, can be reduced accordingly as thedegree of complementarity desired is decreased, and further, dependingupon the G-C or A-T richness of any area wherein variability is searchedfor. Likewise, stringency of hybridization and washing, if desired, canbe increased accordingly as homology desired is increased, and further,ID depending upon the G-C or A-T richness of any area wherein highhomology is desired, all as known in the art.

Another way to define selective hybridization is by looking at theamount (percentage) of one of the nucleic acids bound to the othernucleic acid. For example, in some embodiments selective hybridizationconditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid isbound to the non-limiting nucleic acid. Typically, the non-limitingprimer is in for example, 10 or 100 or 1000 fold excess. This type ofassay can be performed at under conditions where both the limiting andnon-limiting primer are for example, 10 fold or 100 fold or 1000 foldbelow their k_(d), or where only one of the nucleic acid molecules is 10fold or 100 fold or 1000 fold or where one or both nucleic acidmolecules are above their k_(d).

Another way to define selective hybridization is by looking at thepercentage of primer that gets enzymatically manipulated underconditions where hybridization is required to promote the desiredenzymatic manipulation. For example, in some embodiments selectivehybridization conditions would be when at least about, 60, 65, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer isenzymatically manipulated under conditions which promote the enzymaticmanipulation, for example if the enzymatic manipulation is DNAextension, then selective hybridization conditions would be when atleast about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100percent of the primer molecules are extended. Preferred conditions alsoinclude those suggested by the manufacturer or indicated in the art asbeing appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety ofmethods herein disclosed for determining the level of hybridizationbetween two nucleic acid molecules. It is understood that these methodsand conditions may provide different percentages of hybridizationbetween two nucleic acid molecules, but unless otherwise indicatedmeeting the parameters of any of the methods would be sufficient. Forexample if 80% hybridization was required and as long as hybridizationoccurs within the required parameters in any one of these methods it isconsidered disclosed herein.

It is understood that those of skill in the art understand that if acomposition or method meets any one of these criteria for determininghybridization either collectively or singly it is a composition ormethod that is disclosed herein.

3. Nucleic Acids

There are a variety of molecules disclosed herein that are nucleic acidbased, including for example the nucleic acids that encode, for example,the isolated D4 desaturase and D5 elongase as well as any other proteinsdisclosed herein, as well as various functional nucleic acids. Thedisclosed nucleic acids are made up of for example, nucleotides,nucleotide analogs, or nucleotide substitutes. Non-limiting examples ofthese and other molecules are discussed herein. It is understood thatfor example, when a vector is expressed in a cell, that the expressedmRNA will typically be made up of A, C, G, and U. Likewise, it isunderstood that it for example, an antisense molecule is introduced intoa cell or cell environment through for example exogenous delivery, it isadvantageous that the antisense molecule be made up of nucleotideanalogs that reduce the degradation of the antisense molecule in thecellular environment.

a) Nucleotides and Related Molecules

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Anon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate).

A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to nucleotides are well known in the art and would includefor example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, and 2-aminoadenine as well as modifications atthe sugar or phosphate moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety. Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553-6556).

A Watson-Crick interaction is at least one interaction with theWatson-Crick face of a nucleotide, nucleotide analog, or nucleotidesubstitute. The Watson-Crick face of a nucleotide, nucleotide analog, ornucleotide substitute includes the C2, N1, and C6 positions of a purinebased nucleotide, nucleotide analog, or nucleotide substitute and theC2, N3, C4 positions of a pyrimidine based nucleotide, nucleotideanalog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on theHoogsteen face of a nucleotide or nucleotide analog, which is exposed inthe major groove of duplex DNA. The Hoogsteen face includes the N7position and reactive groups (NH2 or O) at the C6 position of purinenucleotides.

b) Sequences

There are a variety of sequences related to, for example, the isolatedD4 desaturase and D5 elongase as well as any other protein disclosedherein that are disclosed on Genbank, and these sequences and others areherein incorporated by reference in their entireties as well as forindividual subsequences contained therein.

A variety of sequences are provided herein and these and others can befound in Genbank, at www.pubmed.gov. Those of Skill in the artunderstand how to resolve sequence discrepancies and differences and toadjust the compositions and methods relating to a particular sequence toother related sequences. Primers and/or probes can be designed for anysequence given the information disclosed herein and known in the art.

c) Primers and Probes

Disclosed are compositions including primers and probes, which arecapable of interacting with the genes disclosed herein. In certainembodiments the primers are used to support DNA amplification reactions.Typically the primers will be capable of being extended in a sequencespecific manner. Extension of a primer in a sequence specific mannerincludes any methods wherein the sequence and/or composition of thenucleic acid molecule to which the primer is hybridized or otherwiseassociated directs or influences the composition or sequence of theproduct produced by the extension of the primer. Extension of the primerin a sequence specific manner therefore includes, but is not limited to,PCR, DNA sequencing, DNA extension, DNA polymerization, RNAtranscription, or reverse transcription. Techniques and conditions thatamplify the primer in a sequence specific manner are preferred. Incertain embodiments the primers are used for the DNA amplificationreactions, such as PCR or direct sequencing. It is understood that incertain embodiments the primers can also be extended using non-enzymatictechniques, where for example, the nucleotides or oligonucleotides usedto extend the primer are modified such that they will chemically reactto extend the primer in a sequence specific manner. Typically thedisclosed primers hybridize with the nucleic acid or region of thenucleic acid or they hybridize with the complement of the nucleic acidor complement of a region of the nucleic acid.

d) Functional Nucleic Acids.

Functional nucleic acids are nucleic acid molecules that have a specificfunction, such as binding a target molecule or catalyzing a specificreaction. Functional nucleic acid molecules can be divided into thefollowing categories, which are not meant to be limiting. For example,functional nucleic acids include antisense molecules, aptamers,ribozymes, triplex forming molecules, and external guide sequences. Thefunctional nucleic acid molecules can act as affectors, inhibitors,modulators, and stimulators of a specific activity possessed by a targetmolecule, or the functional nucleic acid molecules can possess a de novoactivity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with the mRNA of the isolated D4 desaturaseand D5 elongase or the genomic DNA of the isolated D4 desaturase and D5elongase or they can interact with the polypeptide or fragments of theisolated D4 desaturase and D5 elongase. Often functional nucleic acidsare designed to interact with other nucleic acids based on sequencehomology between the target molecule and the functional nucleic acidmolecule. In other situations, the specific recognition between thefunctional nucleic acid molecule and the target molecule is not based onsequence homology between the functional nucleic acid molecule and thetarget molecule, but rather is based on the formation of tertiarystructure that allows specific recognition to take place.

Antisense molecules are designed to interact with a target nucleic acidmolecule through either canonical or non-canonical base pairing. Theinteraction of the antisense molecule and the target molecule isdesigned to promote the destruction of the target molecule through, forexample, RNAseH mediated RNA-DNA hybrid degradation. Alternatively theantisense molecule is designed to interrupt a processing function thatnormally would take place on the target molecule, such as transcriptionor replication. Antisense molecules can be designed based on thesequence of the target molecule. Numerous methods for optimization ofantisense efficiency by finding the most accessible regions of thetarget molecule exist. Exemplary methods would be in vitro selectionexperiments and DNA modification studies using DMS and DEPC. It ispreferred that antisense molecules bind the target molecule with adissociation constant (k_(d)) less than or equal to 10⁻⁶, 10⁻⁸, 10⁻¹⁰ or10⁻¹². A representative sample of methods and techniques which aid inthe design and use of antisense molecules can be found in the followingnon-limiting list of U.S. Pat. Nos. 5,135,917, 5,294,533, 5,627,158,5,641,754, 5,691,317, 5,780,607, 5,786,138, 5,849,903, 5,856,103,5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095,6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025,198, 6,033,910,6,040,296, 6,046,004, 6,046,319, and 6,057,437.

Aptamers are molecules that interact with a target molecule, preferablyin a specific way. Typically aptamers are small nucleic acids rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Aptamers can bind smallmolecules, such as ATP (U.S. Pat. No. 5,631,146) and theophiline (U.S.Pat. No. 5,580,737), as well as large molecules, such as reversetranscriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No.5,543,293). Aptamers can bind very tightly with k_(d)s from the targetmolecule of less than 10⁻¹² M. It is preferred that the aptamers bindthe target molecule with a k_(d) less than 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹².Aptamers can bind the target molecule with a very high degree ofspecificity. For example, aptamers have been isolated that have greaterthan a 10000 fold difference in binding affinities between the targetmolecule and another molecule that differ at only a single position onthe molecule (U.S. Pat. No. 5,543,293). It is preferred that the aptamerhave a k_(d) with the target molecule at least 10, 100, 1000, 10,000, or100,000 fold lower than the k_(d) with a background binding molecule. Itis preferred when doing the comparison for a polypeptide for example,that the background molecule be a different polypeptide. For example,when determining the specificity of the isolated D4 desaturase and D5elongase aptamers, the background protein could be serum albumin.Representative examples of how to make and use aptamers to bind avariety of different target molecules can be found in the followingnon-limiting list of U.S. Pat. Nos. 5,476,766, 5,503,978, 5,631,146,5,731,424, 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660,5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020,6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698.

Ribozymes are nucleic acid molecules that are capable of catalyzing achemical reaction, either intramolecularly or intermolecularly.Ribozymes are thus catalytic nucleic acid. It is preferred that theribozymes catalyze intermolecular reactions. There are a number ofdifferent types of ribozymes that catalyze nuclease or nucleic acidpolymerase type reactions which are based on ribozymes found in naturalsystems, such as hammerhead ribozymes, (for example, but not limited tothe following U.S. Pat. Nos. 5,334,711, 5,436,330, 5,616,466, 5,633,133,5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288,5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but notlimited to the following U.S. Pat. Nos. 5,631,115, 5,646,031, 5,683,902,5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), andtetrahymena ribozymes (for example, but not limited to the followingU.S. Pat. Nos. 5,595,873 and 5,652,107). There are also a number ofribozymes that are not found in natural systems, but which have beenengineered to catalyze specific reactions de novo (for example, but notlimited to the following U.S. Pat. Nos. 5,580,967, 5,688,670, 5,807,718,and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, andmore preferably cleave RNA substrates. Ribozymes typically cleavenucleic acid substrates through recognition and binding of the targetsubstrate with subsequent cleavage. This recognition is often basedmostly on canonical or non-canonical base pair interactions. Thisproperty makes ribozymes particularly good candidates for targetspecific cleavage of nucleic acids because recognition of the targetsubstrate is based on the target substrates sequence. Representativeexamples of how to make and use ribozymes catalyze a variety ofdifferent reactions can be found in the following non-limiting list ofU.S. Pat. Nos. 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855,5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and6,017,756.

Triplex forming functional nucleic acid molecules are molecules that caninteract with either double-stranded or single-stranded nucleic acid.When triplex molecules interact with a target region, a structure calleda triplex is formed, in which there are three strands of DNA forming acomplex dependant on both Watson-Crick and Hoogsteen base-pairing.Triplex molecules are preferred because they cart bind target regionswith high affinity and specificity. It is preferred that the triplexforming molecules bind the target molecule with a k_(d) less than 10⁻⁶,10⁻⁸, 10⁻¹⁰, or 10⁻¹². Representative examples of how to make and usetriplex forming molecules to bind a variety of different targetmolecules can be found in the following non-limiting list of U.S. Pat.Nos. 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834,185,5,869,246, 5,874,566, and 5,962,426.

External guide sequences (EGSs) are molecules that bind a target nucleicacid molecule forming a complex, and this complex is recognized by RNaseP, which cleaves the target molecule, EGSs can be designed tospecifically target a RNA molecule of choice, RNAse P aids in processingtransfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited tocleave virtually any RNA sequence by using an EGS that causes the targetRNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 byYale, and Forster and Altman, Science 238:407-409 (1990)).

Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can beutilized to cleave desired targets within eukaryotic cells. (Yuan etal., Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 byYale; WO 95/24489 by Yale; Yuan and Altman, EMBO J 14:159-168 (1995),and Carrara et al., Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)).Representative examples of how to make and use EGS molecules tofacilitate cleavage of a variety of different target molecules be foundin the following non-limiting list of U.S. Pat. Nos. 5,168,053,5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.

4. Nucleic Acid Delivery

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the disclosed nucleic acids can be in theform of naked DNA or RNA, or the nucleic acids can be in a vector fordelivering the nucleic acids to the cells, whereby the antibody-encodingDNA fragment is under the transcriptional regulation of a promoter, aswould be well understood by one of ordinary skill in the art. The vectorcan be a commercially available preparation, such as an adenovirusvector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Deliveryof the nucleic add or vector to cells can be via a variety ofmechanisms. As one example, delivery can be via a liposome, usingcommercially available liposome preparations such as LIPOFECTIN,LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen,Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,Wis.), as well as other liposomes developed according to proceduresstandard in the art. In addition, the disclosed nucleic acid or vectorcan be delivered in vivo by electroporation, the technology for which isavailable from Genetronics, Inc. (San Diego, Calif.) as well as by meansof a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system which can package a recombinant retroviralgenome (see e.g., Pastan et Proc. Natl. Acad. Sci. U.S.A. 85:4486, 1988;Miller et al., Mol. Cell. Biol. 6:2895, 1986). The recombinantretrovirus can then be used to infect and thereby deliver to theinfected cells nucleic add encoding a broadly neutralizing antibody (oractive fragment thereof). The exact method of introducing the alterednucleic acid into mammalian cells is, of course, not limited to the useof retroviral vectors. Other techniques are widely available for thisprocedure including the use of adenoviral vectors (Mitani et al., Hum.Gene Ther. 5:941-948, 1994), adeno-associated viral (AAV) vectors(Goodman et at, Blood 84:1492-1500, 1994), lentiviral vectors (Naidiniet al., Science 272:263-267, 1996), pseudotyped retroviral vectors(Agrawal et al., Exper. Hematol. 24:738-747, 1996). Physicaltransduction techniques can also be used, such as liposome delivery andreceptor-mediated and other endocytosis mechanisms (see, for example,Schwartzenberger et al., Blood 87:472-478, 1996). This disclosedcompositions and methods can be used in conjunction with any of these orother commonly used gene transfer methods.

As one example, if the antibody-encoding nucleic acid is delivered tothe cells of a subject in an adenovirus vector, the dosage foradministration of adenovirus to humans can range from about 10⁷ to 10⁹plaque forming units (pfu) per injection but can be as high as 10¹² pinper injection (Crystal, Hum. Gene Ther. 8:985-1001, 1997; Alvarez andCuriel, Hum. Gene Ther. 8:597-613, 1997). A subject can receive a singleinjection, or, if additional injections are necessary, they can berepeated at six month intervals (or other appropriate time intervals, asdetermined by the skilled practitioner) for an indefinite period and/oruntil the efficacy of the treatment has been established.

Parenteral administration of the nucleic acid or vector, if used, isgenerally characterized by injection. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution of suspension in liquid prior to injection,or as emulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. For additionaldiscussion of suitable formulations and various routes of administrationof therapeutic compounds, see, e.g., Remington: The Science and Practiceof Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company,Easton, Pa. 1995.

5. Expression Systems

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

a) Viral Promoters and Enhancers

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers et al., Nature, 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment Greenway, P. J. et al., Gene 18:355-360 (1982)). Of course, promoters from the host cell or relatedspecies also are useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′ (Lusky,M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to the transcription unit.Furthermore, enhancers can be within an intron (Banerji, J. L. et al.,Cell 33: 729 (1983)) as well as within the coding sequence itself(Osborne, T. F., et al., Mol. Cell Bio. 4: 1293 (1984)). They areusually between 10 and 300 bp in length, and they function in cis.Enhancers function to increase transcription from nearby promoters.Enhancers also often contain response elements that mediate theregulation of transcription. Promoters can also contain responseelements that mediate the regulation of transcription. Enhancers oftendetermine the regulation of expression of a gene. While many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein and insulin), typically one will use an enhancer from aeukaryotic cell virus for general expression. Preferred examples are theSV40 enhancer on the late side of the replication origin (bp 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

The promoter and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTR.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contains a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

b) Markers

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. Coli lacZ gene, which encodes β-galactosidase,and green fluorescent protein.

In some embodiments the marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: CHO DHFR-cells and mouse LTK-cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employbacterial Qenes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuramycin.

6. Peptides

a) Protein Variants

As discussed herein there are numerous variants of the isolated D4desaturase and D5 elongase proteins that are known and hereincontemplated. In addition, to the known functional the isolated D4desaturase and D5 elongase strain variants there are derivatives of theisolated D4 desaturase and D5 elongase proteins which also function inthe disclosed methods and compositions. Protein variants and derivativesare well understood to those of skill in the art and in can involveamino acid sequence modifications. For example, amino acid sequencemodifications typically fall into one or more of three classes:substitutional, insertional or deletional variants. Insertions includeamino and/or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily will be smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of one to four residues.Immunogenic fusion protein derivatives, such as those described in theexamples, are made by fusing a polypeptide sufficiently large to conferimmunogenicity to the target sequence by cross-linking in vitro or byrecombinant cell culture transformed with DNA encoding the fusion.Deletions are characterized by the removal of one or more amino acidresidues from the protein sequence. Typically, no more than about from 2to 6 residues are deleted at any one site within the protein molecule.These variants ordinarily are prepared by site specific mutagenesis ofnucleotides in the DNA encoding the protein, thereby producing DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, forexample M13 primer mutagenesis and PCR mutagenesis. Amino acidsubstitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingTables 2 and 3 and are referred to as conservative substitutions.

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table3, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or amyl; (b) a cysteine orproline is substituted for or by) an other residue; (c) a residue havingan electropositive side chain, e.g., lysyl, arginyl, or histidyl, issubstituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

TABLE 2 Amino Acid Abbreviations Amino Acid Abbreviations alanine Ala Aarginine Arg R asparagine Asn N aspartic acid Asp D cysteine Cys Cglutamic acid Glu E glutamine Gln K glycine Gly G histidine His Hisolelucine Ile I leucine Leu L lysine Lys K phenylalanine Phe F prolinePro P serine Ser S threonine Thr T tyrosine Tyr Y tryptophan Trp Wvaline Val V methionine Met M

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the mosaicpolypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

TABLE 3 Amino Acid Substitutions Original Residue Exemplary ConservativeSubstitutions, others are known in the art. Ala; ser Arg; lys, gln Asn;gln; his Asp; glu Cys; ser Gln; asn, lys Glu; asp Gly; pro His; asn; glnIle; leu; val Leu; ile; val Lys; arg; gln; Met; Leu; ile Phe; met; leu;tyr Ser; thr Thr; ser Trp; tyr Tyr; trp; phe Val; ile; leu

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

It is understood that one way to define the variants and derivatives ofthe disclosed proteins herein is through defining the variants andderivatives in terms of homology/identity to specific known sequences.For example, SEQ ID NO: 15 sets forth a particular sequence of a D5elongase and SEQ ID NO:26 sets forth a particular sequence of a D4desaturase protein. Specifically disclosed are variants of these andother proteins herein disclosed which have at least, 70% or 75% or 80%or 85% or 90% or 95% homology to the stated sequence. Those of skill inthe art readily understand how to determine the homology of twoproteins. For example, the homology can be calculated after aligning thetwo sequences so that the homology is at its highest level. Specificallydisclosed are sequences having greater than 96% identity and 89%identity to SEC) ID NOs: 15 and 26.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

It is understood that the description of conservative mutations andhomology can be combined together in any combination, such asembodiments that have at least 70% homology to a particular sequencewherein the variants are conservative mutations.

As this specification discusses various proteins and protein sequencesit is understood that the nucleic acids that can encode those proteinsequences are also disclosed. This would include all degeneratesequences related to a specific protein sequence, i.e. all nucleic acidshaving a sequence that encodes one particular protein sequence as wellas all nucleic acids, including degenerate nucleic acids, encoding thedisclosed variants and derivatives of the protein sequences. Thus, whileeach particular nucleic acid sequence may not be written out herein, itis understood that each and every sequence is in fact disclosed anddescribed herein through the disclosed protein sequence. For example,one of the many nucleic acid sequences that can encode the proteinsequence set forth in SEP ID NO:15 is set forth in SEP ID NO:14. It isalso understood that while no amino acid sequence indicates whatparticular DNA sequence encodes that protein within an organism, whereparticular variants of a disclosed protein are disclosed herein, theknown nucleic acid sequence that encodes that protein in the particularstrain from which that protein arises is also known and herein disclosedand described.

It is understood that there are numerous amino acid and peptide analogswhich can be incorporated into the disclosed compositions. For example,there are numerous D amino acids or amino acids which have a differentfunctional substituent then the amino acids shown in Table 2 and Table3. The opposite stereo isomers of naturally occurring peptides aredisclosed, as well as the stereo isomers of peptide analogs. These aminoacids can readily be incorporated into polypeptide chains by chargingtRNA molecules with the amino acid of choice and engineering geneticconstructs that utilize, for example, amber codons, to insert the analogamino acid into a peptide chain in a site specific way (Thorson et al.,Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion inBiotechnology, 3:348-354 (1992); Ibba, Biotechnology & GeneticEngineering Reviews 13:197-216 (1995), Cahill et at, TIBS,14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba andHennecke. Bio/technology, 12:678-682 (1994) all of which are hereinincorporated by reference at least for material related to amino acidanalogs).

Molecules can be produced that resemble peptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH═CH— (cis and trans), —COOH₂—, —CH(OH)CH₂—, and —CHH₂SO— (These andothers can be found in Spatola, F. in Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker,New York, p. 267 (1983); Spatola, A, F., Vega Data (March 1983), Vol. 1,Issue 3, Peptide Backbone Modifications (general review); Morley, TrendsPharm. Sci (1980) pp. 463-468; Hudson, D. et al., Int Pept Prot Res14:177-185 (1979) (—CH₂NH—, CH₂CH₂—); Spatola et al. Life Sci38:1243-1249 (1986) (—CHH₂—S); Hann J. Chem. Soc Perkin Trans. I 307-314(1982) (—CH—CH—, cis and trans); Almquist et al. J. Med. Chem.23:1392-1398 (1980) (—COCH₂—); Jennings-White et al. Tetrahedron Lett23:2533 (1982) (—COCH₂—); Szelke et al, European Appln, EP 45665 CA(1982): 97:39405 (1982) (—CH(OH)CH₂—); Holladay et al. Tetrahedron. Lett24:4401-4404 (1983) (—C(OH)CH₂—); and Hruby Life Sci 31:189-199 (1982)(—CH₂—S—); each of which is incorporated herein by reference. Aparticularly preferred non-peptide linkage is —CH₂NH—. It is understoodthat peptide analogs can have more than one atom between the bond atoms,such as b-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and analogs and peptide analogs often have enhancedor desirable properties, such as, more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers.

D-amino acids can be used to generate more stable peptides, because Damino acids are not recognized by peptidases and such. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g. D-lysine in place of L-lysine) canbe used to generate more stable peptides, Cysteine residues can be usedto cyclize or attach two or more peptides together. This can bebeneficial to constrain peptides into particular conformations. (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference).

7. Antibodies

(1) Antibodies Generally

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of those immunoglobulin molecules, and human orhumanized versions of immunoglobulin molecules or fragments thereof, aslong as they are chosen for their ability to interact with the isolatedD4 desaturases and D5 elongases such that they can be identified, bound,purified, or have altered activity. Antibodies that bind the disclosedregions of the D4 desaturases and D5 elongases are also disclosed. Theantibodies can be tested for their desired activity using the in vitroassays described herein, or by analogous methods, after which their invivo therapeutic and/or prophylactic activities are tested according toknown clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies, theindividual antibodies within the population are identical except forpossible naturally occurring mutations that may be present in a smallsubset of the antibody molecules. The monoclonal antibodies hereinspecifically include “chimeric” antibodies in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, as long as they exhibit the desired antagonisticactivity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl.Acad. Sci. USA, 81:6851-6855 (1984)).

The disclosed monoclonal antibodies can be made using any procedurewhich produces mono clonal antibodies. For example, disclosed monoclonalantibodies can be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse or other appropriate host animal is typically immunizedwith an immunizing agent to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro, e.g., using, the HIV Env-CD4-co-receptor complexes describedherein.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNAencoding the disclosed monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Libraries of antibodies oractive antibody fragments can also be generated and screened using phagedisplay techniques, e.g., as described in U.S. Pat. No. 5,804,440 toBurton et al. and U.S. Pat. No. 6,096,411 to Barbas et al.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fe fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross-linking antigen.

The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the antibody or antibody fragment is notsignificantly altered or impaired compared to the non-modified antibodyor antibody fragment. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, to alter its secretorycharacteristics, etc. In any case, the antibody or antibody fragmentmust possess a bioactive property, such as specific binding to itscognate antigen. Functional or active regions of the antibody orantibody fragment may be identified by mutagenesis of a specific regionof the protein, followed by expression and testing of the expressedpolypeptide. Such methods are readily apparent to a skilled practitionerin the art and can include site-specific mutagenesis of the nucleic acidencoding the antibody or antibody fragment. (Zoller, M. S. Curr. Opin.Biotechnol. 3:348-354, 1992).

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody. Many non-human antibodies(e.g., those derived from mice, rats, or rabbits) are naturallyantigenic in humans, and thus can give rise to undesirable immuneresponses when administered to humans. Therefore, the use of human orhumanized antibodies in the methods serves to lessen the chance that anantibody administered to a human will evoke an undesirable immuneresponse.

(2) Human Antibodies

The disclosed human antibodies can be prepared using any technique.Examples of techniques for human monoclonal antibody production includethose described by Cole et al. (Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol.,147(1):86-95, 1991). Human antibodies (and fragments thereof) can alsobe produced using Phage display libraries (Hoogenboom et al., J. Mol.Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991).

The disclosed human antibodies can also be obtained from transgenicanimals. For example, transgenic, mutant mice that are capable ofproducing a full repertoire of human antibodies, in response toimmunization, have been described (see, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)).Specifically, the homozygous deletion of the antibody heavy chainjoining region (J(H)) gene in these chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production, andthe successful transfer of the human germ-line antibody gene array intosuch germ-line mutant mice results in the production of human antibodiesupon antigen challenge. Antibodies having the desired activity areselected using Env-CD4-co-receptor complexes as described herein.

(3) Humanized Antibodies

Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of art antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as anFv, Fab, Fab′, or other antigen-binding portion of an antibody) whichcontains a portion of an antigen binding site from a non-human (donor)antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen). In some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.In practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fe), typically that of a human antibody (Jones et Nature, 321:522-525(1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr.Opin. Struct. Biol., 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones et al., Nature, 321:522-525(1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332(Hoogenboom et at), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No.5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.),U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377(Morgan et al.).

(4) Administration of Antibodies

Administration of the antibodies can be done as disclosed herein.Nucleic acid approaches for antibody delivery also exist. The antibodiesand antibody fragments can also be administered to patients or subjectsor cells as a nucleic acid preparation (e.g., DNA or RNA) that encodesthe antibody or antibody fragment, such that the patient's or subject'sown cells take up the nucleic acid and produce and secrete the encodedantibody or antibody fragment. The delivery of the nucleic acid can beby any means, as disclosed herein, for example.

8. Pharmaceutical Carriers/Delivery of Pharmaceutical Products

As described above, the compositions can also be administered in vivo ina pharmaceutically acceptable carrier. By “pharmaceutically acceptable”is meant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to a subject, along with thenucleic acid or vector, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,including topical intranasal administration or administration byinhalant. As used herein, “topical intranasal administration” meansdelivery of the compositions into the nose and nasal passages throughone or both of the nares and can comprise delivery by a sprayingmechanism or droplet mechanism, or through aerosolization of the nucleicacid or vector. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the allergic disorder being treated, the particular nucleicacid or vector used, its mode of administration and the like. Thus, itis not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451. (1991); Bagshawe, K. D., Br. J Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells inviva. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

a) Pharmaceutically Acceptable Carriers

The compositions, including antibodies, can be used therapeutically incombination with a pharmaceutically acceptable carrier.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Further carriers include sustained release preparationssuch as semipermeable matrices of solid hydrophobic polymers containingthe antibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the mute of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedantibodies can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

b) Therapeutic Uses

Effective dosages and schedules for administering the compositions maybe determined empirically, and making such determinations is within theskill in the art. The dosage ranges for the administration of thecompositions are those large enough to produce the desired effect inwhich the symptoms disorder are effected. The dosage should not be solarge as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient, route of administration, or whether other drugs areincluded in the regimen, and can be determined by one of skill in theart. The dosage can be adjusted by the individual physician in the eventof any counterindications. Dosage can vary, and can be administered inone or more dose administrations daily, for one or several days.Guidance can be found in the literature for appropriate dosages forgiven classes of pharmaceutical products. For example, guidance inselecting appropriate doses for antibodies can be found in theliterature on therapeutic uses of antibodies, e.g., Handbook ofMonoclonal Antibodies, Ferrone et al., eds., Noges Publications, ParkRidge. N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies inHuman Diagnosis and Therapy, Haber et al., eds., Raven Press, New York(1977) pp. 365-389. A typical daily dosage of the antibody used alonemight range from about 1 μg/kg to up to 100 mg/kg of body weight or moreper day, depending on the factors mentioned above.

Other molecules that interact with the isolated D4 desaturases and D5elongases which do not have a specific pharmaceutical function but whichmay be used for tracking changes within cellular chromosomes or for thedelivery of diagnostic tools for example can be delivered in wayssimilar to those described for the pharmaceutical products.

9. Chips and Micro Arrays

Disclosed are chips where at least one address is the sequences or partof the sequences set forth in any of the nucleic acid sequencesdisclosed herein. Also disclosed are chips where at least one address isthe sequences or portion of sequences set forth in any of the peptidesequences disclosed herein.

Also disclosed are chips where at least one address is a variant of thesequences or part of the sequences set forth in any of the nucleic acidsequences disclosed herein. Also disclosed are chips where at least oneaddress is a variant of the sequences or portion of sequences set forthin any of the peptide sequences disclosed herein.

10. Computer Readable Mediums

It is understood that the disclosed nucleic acids and proteins can berepresented as a sequence consisting of the nucleotides of amino acids.There are a variety of ways to display these sequences, for example thenucleotide guanosine can be represented by G or g. Likewise the aminoacid valine can be represented by Val or V. Those of skill in the artunderstand how to display and express any nucleic acid or proteinsequence in any of the variety of ways that exist, each of which isconsidered herein disclosed. Specifically contemplated herein is thedisplay of these sequences on computer readable mediums, such as,commercially available floppy disks, tapes, chips, hard drives, compactdisks, and video disks, or other computer readable mediums. Alsodisclosed are the binary code representations of the disclosedsequences. Those of skill in the art understand what computer readablemediums. Thus, computer readable mediums on which the nucleic acids orprotein sequences are recorded, stored, or saved.

Disclosed are computer readable mediums comprising the sequences andinformation regarding the sequences set forth herein.

11. Compositions Identified by Screening with Disclosed CompositionsCombinatorial Chemistry

a) Combinatorial Chemistry

The disclosed compositions can be used as targets for any combinatorialtechnique to identify molecules or macromolecular molecules thatinteract with the disclosed compositions in a desired way. The nucleicacids, peptides, and related molecules disclosed herein can be used astargets for the combinatorial approaches. Also disclosed are thecompositions that are identified through combinatorial techniques orscreening techniques in which the compositions disclosed in for exampleSEQ ID NOS:14, 15, 25, or 26 or portions thereof, are used as the targetin a combinatorial or screening protocol.

It is understood that when using the disclosed compositions incombinatorial techniques or screening methods, molecules, such asmacromolecular molecules, will be identified that have particulardesired properties such as inhibition or stimulation or the targetmolecule's function. The molecules identified and isolated when usingthe disclosed compositions, such as, a fatty acid, are also disclosed.Thus, the products produced using the combinatorial or screeningapproaches that involve the disclosed compositions, such as, fattyacids, are also considered herein disclosed.

155. IL is understood that the disclosed methods for identifyingmolecules that inhibit the interactions between, for example, theisolated D4 desaturases and the D5 don gases can be performed using highthrough put means. For example, putative inhibitors can be identifiedusing Fluorescence Resonance Energy Transfer (FRET) to quickly identifyinteractions. The underlying theory of the techniques is that when twomolecules are close in space, ie, interacting at a level beyondbackground, a signal is produced or a signal can be quenched. Then, avariety of experiments can be performed, including, for example, addingin a putative inhibitor. If the inhibitor competes with the interactionbetween the two signaling molecules, the signals will be removed fromeach other in space, and this will cause a decrease or an increase inthe signal, depending on the type of signal used. This decrease orincreasing signal can be correlated to the presence or absence of theputative inhibitor. Any signaling means can be used. For example,disclosed are methods of identifying an inhibitor of the interactionbetween any two of the disclosed molecules comprising, contacting afirst molecule and a second molecule together in the presence of aputative inhibitor, wherein the first molecule or second moleculecomprises a fluorescence donor, wherein the first or second molecule,typically the molecule not comprising the donor, comprises afluorescence acceptor; and measuring Fluorescence Resonance EnergyTransfer (FRET), in the presence of the putative inhibitor and the inabsence of the putative inhibitor, wherein a decrease in FRET in thepresence of the putative inhibitor as compared to FRET measurement inits absence indicates the putative inhibitor inhibits binding betweenthe two molecules. This type of method can be performed with a cellsystem as well.

Combinatorial chemistry includes but is not limited to all methods forisolating small molecules or macromolecules that are capable of bindingeither a small molecule or another macromolecule, typically in aniterative process. Proteins, oligonucleotides, and sugars are examplesof macromolecules. For example, oligonucleotide molecules with a givenfunction, catalytic or ligand-binding, can be isolated from a complexmixture of random oligonucleotides in what has been referred to as “invitro genetics” (Szostak, TIBS 19:89, 1992). One synthesizes a largepool of molecules bearing random and defined sequences and subjects thatcomplex mixture, for example, approximately 10¹⁵ individual sequences in100 μg of a 100 nucleotide RNA, to some selection and enrichmentprocess. Through repeated cycles of affinity chromatography and PCRamplification of the molecules bound to the ligand on the column,Ellington and Szostak (1990) estimated that 1 in 10¹⁰ RNA moleculesfolded in such a way as to bind a small molecule dyes. DNA moleculeswith such ligand-binding behavior have been isolated as well (Ellingtonand Szostak, 1992; Bock et al, 1992). Techniques aimed at similar goalsexist for small organic molecules, proteins, antibodies and othermacromolecules known to those of skill in the art. Screening sets ofmolecules for a desired activity whether based on small organiclibraries, oligonucleotides, or antibodies is broadly referred to ascombinatorial chemistry. Combinatorial techniques are particularlysuited for defining binding interactions between molecules and forisolating molecules that have a specific binding activity, often calledaptamers when the macromolecules are nucleic acids.

There are a number of methods for isolating proteins which either havede novo activity or a modified activity. For example, phage displaylibraries have been used to isolate numerous peptides that interact witha specific target. (See for example, U.S. Pat. Nos. 6,031,071;5,824,520; 5,596,079; and 5,565,332 which are herein incorporated byreference at least for their material related to phage display andmethods relate to combinatorial chemistry)

A preferred method for isolating proteins that have a given function isdescribed by Roberts and Szostak (Roberts R. W. and Szostak J. W, Proc.Natl. Acad. Sci. USA, 94(23)12997-302 (1997). This combinatorialchemistry method couples the functional power of proteins and thegenetic power of nucleic acids. An RNA molecule is generated in which apuromycin molecule is covalently attached to the 3′-end of the RNAmolecule. An in vitro translation of this modified RNA molecule causesthe correct protein, encoded by the RNA to be translated. In addition,because of the attachment of the puromycin, a peptidyl acceptor whichcannot be extended, the growing peptide chain is attached to thepuromycin which is attached to the RNA. Thus, the protein molecule isattached to the genetic material that encodes it. Normal in vitroselection procedures can now be done to isolate functional peptides.Once the selection procedure for peptide function is completetraditional nucleic acid manipulation procedures are performed toamplify the nucleic acid that codes for the selected functionalpeptides. After amplification of the genetic material, new RNA istranscribed with puromycin at the 3′-end, new peptide is translated andanother functional round of selection is performed. Thus, proteinselection can be performed in an iterative manner just like nucleic acidselection techniques. The peptide which is translated is controlled bythe sequence of the RNA attached to the puromycin. This sequence can beanything from a random sequence engineered for optimum translation (i.e.no stop codons etc.) or it can be a degenerate sequence of a known RNAmolecule to look for improved or altered function of a known peptide.The conditions for nucleic acid amplification and in vitro translationare well known to those of ordinary skill in the art and are preferablyperformed as in Roberts and Szostak (Roberts R. W. and Szostak J. W.Proc. Natl. Acad. Sci. USA, 94(23)12997-302 (1997)).

Another preferred method for combinatorial methods designed to isolatepeptides is described in Cohen et at (Cohen B. A., et al., Proc. Natl.Acad. Sci. USA 95(24):14272-7 (1998)). This method utilizes and modifiestwo-hybrid technology. Yeast two-hybrid systems are useful for thedetection and analysis of protein:protein interactions. The two-hybridsystem, initially described in the yeast Saccharomyces cerevisiae, is apowerful molecular genetic technique for identifying new regulatorymolecules, specific to the protein of interest (Fields and Song, Nature340:245-6 (1989)). Cohen et al., modified this technology so that novelinteractions between synthetic or engineered peptide sequences could beidentified which hind a molecule of choice. The benefit of this type oftechnology is that the selection is done in an intracellularenvironment. The method utilizes a library of peptide molecules thatattached to an acidic activation domain. A peptide of choice, forexample a portion of the isolated D4 desaturases or D5 elongases isattached to a DNA binding domain of a transcriptional activationprotein, such as Gal 4. By performing the Two-hybrid technique on thistype of system, molecules that bind the portion of the isolated D4desaturases or D5 elongases can be identified.

Using methodology well known to those of skill in the art, incombination with various combinatorial libraries, one can isolate andcharacterize those small molecules or macromolecules, which bind to orinteract with the desired target. The relative binding affinity of thesecompounds can be compared and optimum compounds identified usingcompetitive binding studies, which are well known to those of skill inthe art.

Techniques for making combinatorial libraries and screeningcombinatorial libraries to isolate molecules which bind a desired targetare well known to those of skill in the art. Representative techniquesand methods can be found in but are not limited to U.S. Pat. Nos.5,084,824, 5,288,514, 5,449,754, 5,506,337, 5,539,083, 5,545,568,5,556,762, 5,565,324, 5,565332, 5,573,905, 5,618,825, 5,619,680,5,627,210, 5,646,285, 5,663,046, 5,670,326, 5,677,195, 5,683,899,5,688,696, 5,688,997, 5,698,685, 5,712,146, 5,721,099, 5,723,598,5,741,713, 5,792,431, 5,807,683, 5,807,754, 5,821,130, 5,831,014,5,834,195, 5,834,318, 5,834,588, 5,840,500, 5,847,150, 5,856,107,5,856,496, 5,859,190, 5,864,010, 5,874,443, 5,877,214, 5,880,972,5,886,126, 5,886,127, 5,891,737, 5,916,899, 5,919,955, 5,925,527,5,939,268, 5,942,387, 5,945,070, 5,948,696, 5,958,702, 5,958,792,5,962,337, 5,965,719, 5,972,719, 5,976,894, 5,980,704, 5,985,356,5,999,086, 6,001,579, 6,004,617, 6,008,321, 6,017,768, 6,025,371,6,030,917, 6,040,193, 6,045,671, 6,045,755, 6,060,596, and 6,061,636.

Combinatorial libraries can be made from a wide array of molecules usinga number of different synthetic techniques. For example, librariescontaining fused 2,4-pyrimidinediones (U.S. Pat. No. 6,025,371)dihydrobenzopyrans (U.S. Pat. Nos. 6,017,768 and 5,821,130), amidealcohols (U.S. Pat. No. 5,976,894), hydroxy-amino acid amides (U.S. Pat.No. 5,972,719) carbohydrates (U.S. Pat. No. 5,965,719),1,4-benzodiazepin-2,5-diones (U.S. Pat. No. 5,962,337), cyclics (U.S.Pat. No. 5,958,792), biaryl amino acid amides (U.S. Pat. No. 5,948,696),thiophenes (U.S. Pat. No. 5,942,387), tricyclic Tetrahydroquinolines(U.S. Pat. No. 5,925,527), benzofurans (U.S. Pat. No. 5,919,955),isoquinolines (U.S. Pat. No. 5,916,899), hydantoin and thiohydantoin(U.S. Pat. No. 5,859,190), indoles (U.S. Pat. No. 5,856,496),imidazol-pyrido-indole and imidazol-pyrido-benzothiophenes (U.S. Pat.No. 5,856,107) substituted 2-methylene-2, 3-dihydrothiazoles (U.S. Pat.No. 5,847,150), quinolines (U.S. Pat. No. 5,840,500), PNA (U.S. Pat. No.5,831,014), containing tags (U.S. Pat. No. 5,721,099), polyketides (U.S.Pat. No. 5,712,146), morpholino-subunits (U.S. Pat. Nos. 5,698,685 and5,506,337), sulfamides (U.S. Pat. No. 5,618,825), and benzodiazepines(U.S. Pat. No. 5,288,514).

As used herein combinatorial methods and libraries included traditionalscreening methods and libraries as well as methods and libraries used initerative processes.

b) Computer Assisted Drug Design

The disclosed compositions can be used as targets for any molecularmodeling technique to identify either the structure of the disclosedcompositions or to identify potential or actual molecules, such as smallmolecules, which interact in a desired way with the disclosedcompositions. The nucleic acids, peptides, and related moleculesdisclosed herein can be used as targets in any molecular modelingprogram or approach.

It is understood that when using the disclosed compositions in modelingtechniques, molecules, such as macromolecular molecules, will beidentified that have particular desired properties such as inhibition orstimulation or the target molecule's function. The molecules identifiedand isolated when using the disclosed compositions, such as, theisolated D4 desaturases or D5 elongases, are also disclosed. Thus, theproducts produced using the molecular modeling approaches that involvethe disclosed compositions, such as, the isolated D4 desaturases or D5elongases, are also considered herein disclosed.

Thus, one way to isolate molecules that bind a molecule of choice isthrough rational design. This is achieved through structural informationand computer modeling. Computer modeling technology allows visualizationof the three-dimensional atomic structure of a selected molecule and therational design of new compounds that will interact with the molecule.The three-dimensional construct typically depends on data from x-raycrystallographic analyses or NMR imaging of the selected molecule. Themolecular dynamics require force field data. The computer graphicssystems enable prediction of how a new compound will link to the targetmolecule and allow experimental manipulation of the structures of thecompound and target molecule to perfect binding specificity. Predictionof what the molecule-compound interaction will be when small changes aremade in one or both requires molecular mechanics software andcomputationally intensive computers, usually coupled with user-friendly,menu-driven interfaces between the molecular design program and theuser.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms, Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific proteins, such as Rotivinen, et al., 1988 Acta PharmaceuticaFennica 97, 159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988);McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol. Toxiciol. 29, 111-122;Perry and Davies, QSAR: Quantitative Structure-Activity Relationships inDrug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989Proc. R. Soc. Lond. 236, 125-140 and 141-162; and, with respect to amodel enzyme for nucleic acid components, Askew, et al., 1989 J. Am.Chem. Soc. 111, 1082-1090. Other computer programs that screen andgraphically depict chemicals are available from companies such asBioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario,Canada, and Hypercube, Inc., Cambridge, Ontario. Although these areprimarily designed for application to drugs specific to particularproteins, they can be adapted to design of molecules specificallyinteracting with specific regions of DNA or RNA, once that region isidentified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichalter substrate binding or enzymatic activity.

12. Kits

Disclosed herein are kits that are drawn to reagents that can be used inpracticing the methods disclosed herein. The kits can include anyreagent or combination of reagent discussed herein or that would beunderstood to be required or beneficial in the practice of the disclosedmethods. For example, the kits could include primers to perform theamplification reactions discussed in certain embodiments of the methods,as well as the buffers and enzymes required to use the primers asintended.

13. Compositions with Similar Functions

It is understood that the compositions disclosed herein have certainfunctions, such as enzymatic functions disclosed. Disclosed herein arecertain structural requirements for performing the disclosed functions,and it is understood that there are a variety of structures which canperform the same function which are related to the disclosed structures,and that these structures will ultimately achieve the same result, forexample stimulation or inhibition of the enzymatic function.

C. Methods of Making the Compositions

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted.

1. Nucleic Acid Synthesis

For example, the nucleic acids, such as, the oligonucleotides to be usedas primers can be made using standard chemical synthesis methods or canbe produced using enzymatic methods or any other known method. Suchmethods can range from standard enzymatic digestion followed bynucleotide fragment isolation (see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) topurely synthetic. methods, for example, by the cyanoethylphosphoramidite method using a Milligen or Beckman System 1Plus DNAsynthesizer (for example, Model 8700 automated synthesizer ofMilligen-Bioseareh, Burlington, Mass. or ABI Model 380B). Syntheticmethods useful for making oligonucleotides are also described by Ikutaal., Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester andphosphite-triester methods), and Narang et al., Methods Enzymol.,65:610-620 (1980), (phosphotriester method). Protein nucleic acidmolecules can be made using known methods such as those described byNielsen et al., Bioconjug. Chem. 5:3-7 (1994).

2. Peptide Synthesis

One method of producing the disclosed proteins, such as SEQ ID NO:23, isto link two or more peptides or polypeptides together by proteinchemistry techniques. For example, peptides or polypeptides can bechemically synthesized using currently available laboratory equipmentusing either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc(tert-butyloxycarbonoyl) chemistry (Applied Biosystems, Inc., FosterCity, Calif.), One skilled in the art can readily appreciate that apeptide or polypeptide corresponding to the disclosed proteins, forexample, can be synthesized by standard chemical reactions. For example,a peptide or polypeptide can be synthesized and not cleaved from itssynthesis resin whereas the other fragment of a peptide or protein canbe synthesized and subsequently cleaved from the resin, thereby exposinga terminal group which is functionally blocked on the other fragment. Bypeptide condensation reactions, these two fragments can be covalentlyjoined via a peptide bond at their carboxyl and amino termini,respectively, to form an antibody, or fragment thereof. (Grant G A(1992) Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y.(1992); Bodansky M and Trost B., Ed. (1993) Principles of PeptideSynthesis. Springer-Verlag Inc., NY (which is herein incorporated byreference at least for material related to peptide synthesis).Alternatively, the peptide or polypeptide is independently synthesizedin vivo as described herein. Once isolated, these independent peptidesor polypeptides may be linked to form a peptide or fragment thereof viasimilar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779(1994)). The first step is the chemoselective reaction of an unprotectedsynthetic peptide-thioester with another unprotected peptide segmentcontaining an amino-terminal Cys residue to give a thioester-linkedintermediate as the initial covalent product. Without a change in thereaction conditions, this intermediate undergoes spontaneous, rapidintramolecular reaction to form a native peptide bond at the ligationsite (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I etal., J. Biol. Chem. 269:16075 (1994); Clark-Lewis et al., Biochemistry,30:3128 (1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer, M et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (de Lisle Milton R C et al.,Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267 (1992)).

3. Process Claims for Making the Compositions

Disclosed are processes for making the compositions as well as makingthe intermediates leading to the compositions. For example, disclosedare nucleic acids in SEQ ID NOs:14 and 25. There are a variety ofmethods that can be used for making these compositions, such assynthetic chemical methods and standard molecular biology methods. It isunderstood that the methods of making these and the other disclosedcompositions are specifically disclosed.

Disclosed are nucleic acid molecules produced by the process comprisinglinking in an operative way a nucleic acid comprising the sequence setforth in SEQ ID NO:14 and 25, for example, and a sequence controllingthe expression of the nucleic acid.

Also disclosed are nucleic acid molecules produced by the processcomprising linking in an operative way a nucleic acid moleculecomprising a sequence having 80% identity to a sequence set forth in SEQID NO:14 and 25, for example, and a sequence controlling the expressionof the nucleic acid.

Disclosed are nucleic acid molecules produced by the process comprisinglinking in an operative was a nucleic acid molecule comprising asequence that hybridizes under stringent hybridization conditions to asequence set forth SEQ ID NO:14 and 25, for example, and a sequencecontrolling the expression of the nucleic acid.

Disclosed are nucleic acid molecules produced by the process comprisinglinking in an operative way a nucleic acid molecule comprising asequence encoding a peptide set forth in SEQ ID NO:15 and 26, forexample, and a sequence controlling an expression of the nucleic acidmolecule.

Disclosed are nucleic acid molecules produced by the process comprisinglinking in an operative way a nucleic acid molecule comprising asequence encoding a peptide having 80% identity to a peptide set forthin SEQ ID NO:15 and 26, for example, and a sequence controlling anexpression of the nucleic acid molecule.

Disclosed are nucleic acids produced by the process comprising linkingin an operative way a nucleic acid molecule comprising a sequenceencoding a peptide having 80% identity to a peptide set forth in SEQ IDNO:15 and 26, for example, wherein any change from the SEQ ID NO:15 and26, for example, are conservative changes and a sequence controlling anexpression of the nucleic acid molecule.

Disclosed are cells produced by the process of transforming the cellwith any of the disclosed nucleic acids. Disclosed are cells produced bythe process of transforming the cell with any of the non-naturallyoccurring disclosed nucleic acids.

Disclosed are any of the disclosed peptides produced by the process ofexpressing any of the disclosed nucleic acids. Disclosed are any of thenon-naturally occurring disclosed peptides produced by the process ofexpressing any of the disclosed nucleic acids. Disclosed are any of thedisclosed peptides produced by the process of expressing any of thenon-naturally disclosed nucleic acids.

Disclosed are animals produced by the process of transfecting a cellwithin the animal with any of the nucleic acid molecules disclosedherein. Disclosed are animals produced by the process of transfecting acell within the animal any of the nucleic acid molecules disclosedherein, wherein the animal is a mammal. Also disclosed are animalsproduced by the process of transfecting a cell within the animal any ofthe nucleic acid molecules disclosed herein, wherein the mammal ismouse, rat, rabbit, cow, sheep, pig, or primate.

Also disclose are animals produced by the process of adding to theanimal any of the cells disclosed herein.

D. EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

1. Example 1 Design of Degenerate Oligonucleotides for the Isolation ofDesaturase and Elongase from Thraustochytrid ONC-T18

Analysis of the fatty acid composition of Thraustochytrid ONC-T18revealed the presence of considerable amount of longer chain PITA suchas arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA,20:5n-3), adrenic acid (ADA, 22:4n-6, {acute over (ω)}6-docosapentaenoicacid ({acute over (ω)}6-DPA, 22:5n-6), {acute over(ω)}3-docosapentaenoic acid ({acute over (ω)}3-DPAn-3), anddocosahexaenoic acid (DHA, 22:6n-3). Thus it was thought that thisorganism contained an active Δ5-elongase capable of converting ARA toADA or EPA to {acute over (ω)}3-DPA and an active Δ4-desaturase whichdesaturates ADA to {acute over (ω)}6-DPA or {acute over (ω)}3-DPA to DHA(FIG. 1). The goal thus was to attempt to isolate these desaturase andelongase genes from Thraustochytrid ONC-T18, and eventually to verifythe functionality by expression in an alternate host.

To isolate genes encoding functional desaturase enzymes, genomic DNA wasextracted from the organism. Thraustochytrid ONC-T18 cultures were grownin a growth medium (5 g/l yeast extract, 5 g/l peptone, 40 g/lD(+)-glucose, 1.25 ml/l trace elements, 1.25 ml/l vitamins, 40 g/l seasalt; (trace elements: 5 g/l NaH₂PO₄.H₂O, 3.15 g/l FeCl₃.6H₂O, 4.36 g/lNa₂EDTA.2H₂O, 0.6125 mg/l CuSO₄. 5H₂0, 0.05970 g/l Na₂MoO₄.2H₂O, 0.022g/l ZnSO₄.7H₂0, 0.01 g/l CoCl₂.6H₂O, 0.18 MnCl₂.4H₂O, 13 μg/l H₂SeO₃,2.7 mg/l NiSO₄.6H₂O, 1.84 mg/l Na₃VO₄, 1.94 mg/l K₂CrO₄), (vitamins: 1mg/l vitamin B12, 1 mg/l biotin, 0.20 g/l thiamine HCl)) at 26° C. for16-20 hours with constant agitation. The cells were centrifuged 500 rpm,5 min at room temperature in Sorvall Super T21 centrifuge with rotorST-H750 using adapter Sorvall #00436, removed 80% of supernatant,resuspended cells in remaining medium and continued with extraction.Genomic DNA was isolated from cells using Ultraclean Microbial DNAIsolation kit (MO BIO Laboratories, Inc, Solana Beach, Calif.) as permanufacturer's protocol.

The approach taken was to design degenerate oligonucleotides (i.e.,primers) that are conserved in known desaturases. These primers could beused in a PCR reaction to identify a fragment containing the conservedregions in the predicted desaturase genes from Thraustochytrium. Fivesequences were available from Thraustochytrium sp., Thraustochytriumaureum, Thraustochytrium sp. ATCC 34304, Thraustochytrium sp. ATCC21685, and Thraustochytrium sp. FJN-10 (EMBO accession number CS020087,Genbank accession number AF391546, AF391543, AF489589, DQ133575,respectively). The degenerate primers used were as follows using theKodon primer designer software: Primer 4desat308 (Forward) (SEQ ID NO:1)5′-GGRACAGCGASTTTTACAGGG-3′, Primer 4desat1369 (Reverse) (SEQ ID NO:2)5′-GTGCTCAATCTGGTGGTTKAG-3′.

The same approach was taken to design degenerate oligonucleotides (i.e.,primers) that are conserved in known elongases. These primers could beused in a PCR reaction to identify a fragment containing the conservedregions in the predicted elongase genes from Thraustochytrium sp. Twosequences were available from Thraustochytrium sp, and Thraustochytriumaureum (EMBO accession number CS160897 and CS160879, respectively). Thedegenerate primers used were as follows using Kodon primer designersoftware: Primer 5elo202F (Forward) (SEQ ID NO:3)5′-AAGCCYTTCGAGCTCAAGTYC-3′, Primer 5elo768R (Reverse′) (SEQ ID NO:4)5′-GCACGAARAAGTTGCCGAAG-3′. The degeneracy code for the oligonucleotidesequences was: K=G,T, R=A,G, S=G,C, Y=C,T.

2. Example 2 Isolation of Δ5-Elongase Nucleotide Sequences fromThraustochytrium sp. ONC-T18

To isolate the Δ5-elongase gene, PCR amplification was carried out in a50 μl volume containing: 200 ng Thraustochytrium sp. ONC-T18 genomicDNA, 10 μl betaine, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂,0.001% gelatin, 200 μM each deoxyribonucleotide triphosphate, 1 μM ofeach primer and 0.2 unit of Taq DNA Polymerase (Sigma, Oakville,Ontario). Thermocycling was carried out at an annealing temperature of55.7° C., the PCR reaction was resolved on a 0.8% low melt agarose gel,and the band of ˜600 bp was gel purified. PCR product was eluted fromagarose with water and then purified with QIAquick PCR Purification kit(Qiagen, Valencia, Calif.). These DNA fragments were cloned into thepT7Blue-3 Perfectly Blunt Cloning kit (Novagen, San Diego, Calif.) asper manufacturer's specifications. The recombinant plasmids weretransformed into NovaBlue competent cells (Novagen, San Diego, Calif.),and clones were sequenced. One clone was thus isolated that showedsequence homology to previously identified Δ5-elongase. This clone isdescribed as follows: Clone #600-16 (SEQ ID NO:5) was sequenced and thededuced amino acid sequence from 593 bp showed 95% identity withpolyunsaturated fatty acid elongase 1 Thraustochytrium sp. FJN-10 as thehighest scoring match in a BLASTx search.

To isolate the 3′-end, genome walking was carried out using APAgene GOLDGenome Walking kit (BIO S&T, Montreal, Quebec) as per manufacturer'sprotocol, using genomic DNA purified from Thraustochytrium sp. ONC-T18,and oligonucleotides 3′GSPa (SEQ ID NO:6)(5″-CGCTGCGCCCGTACATTACTACCATCCA-3′) and 3′GSPb (SEQ ID NO:7)(5′-GTCGTCCAGTCCGTCTATGAC-3′). Both oligonucleotides were designed basedon the #600-16 fragment of the putative Δ5-elongase. The PCR fragmentswere resolved on 0.8% low melt agarose gel, and the bands of ˜500 to2000 bp were gel purified. PCR products were eluted from agarose withwater and then purified with QIAquick PCR Purification kit (Qiagen,Valencia, Calif.). These DNA fragments were cloned into the pT7Blue-3Perfectly Blunt Cloning kit (Novagen, San Diego, Calif.) as permanufacturer's specifications. The recombinant plasmids were transformedinto NovaBlue competent cells (Novagen, San Diego, Calif.), and cloneswere sequenced. Clone 3′C2-1 (SEQ ID NO:8) contained a 358 bp insertwhich was identified to contain the 3′-end of the putative Δ5-elongasegene based on sequence homology with known Δ5-elongase and the presenceof the ‘TGA’ stop codon.

To isolate the 5′-end, genome walking was carried out using APAgene GOLDGenome Walking kit (BIO S&T, Montreal, Quebec) as per manufacturer'sprotocol, using genomic DNA purified from Thraustochytrium sp. ONC-T18,and oligonucleotides 5′GSPa (SEQ ID NO:9)(5′-CTCGGCACCCTTCTCCATCGGGTTGCCA-3′) and 5′GSPb (SEQ ID NO:10)(5′-GTTGCCAAAGAGCTTGTAGCCGCCGA-3′). Both oligonucleotides were designedbased on the #600-16 fragment of the putative Δ5-elongase. The PCRfragments were resolved on 0.8% low melt agarose gel, and the bands of˜500 to 2000 bp were gel purified. PCR products were doted from agarosewith water and then purified with QIAquick PCR Purification kit (Qiagen,Valencia, Calif.). These DNA fragments were cloned into the pT7Blue-3Perfectly Blunt Cloning kit (Novagen, San Diego, Calif.) as permanufacturer's specifications. The recombinant plasmids were transformedinto NovaBlue competent cells (Novagen, San Diego, Calif.), and cloneswere sequenced. Clone 5′D2-11 (SEQ ID NO:11) was thus obtained thatcontained a 519 bp insert that contained the putative ‘ATG’ start siteof the novel Δ5-elongase. The deduced amino acid sequence of thisfragment, when aligned with known Δ5-elongase showed 89% identity.

This Δ5-elongase gene was isolated in its entirety by PCR amplificationusing, the Thraustochytrium ONC-T18 genomic DNA as a template, and thefollowing oligonucleotides: ONC-T18elo1 (Forward) (SEQ ID NO:12)5′-GCTGATGATGGCCGGGACC-3′, ONC-T18elo1099 (Reverse) (SEQ NO:13)5′-GCTCCACTCGAATTCGTAGCG-3′.

PCR amplification was carried out using in a 50 μl volume: 100 ng of theThraustochytrium ONC-T18 genomic DNA, 25 mM TAPS-HCl, pH 9.3, 50 mM KCl,2 mM MgCl₂, 1 mM β-mercaptoethanol, 1.5 μl DMSO, 200 μM eachdeoxyribonucleotide triphosphate, 0.5 μM of each primer and 1 unitPhusion high-fidelity DNA polymerase (Finnzymes, Espoo, Finland).Thermocycling conditions were as follows: the template was initiallydenatured at 98° C. for 30 sec, followed by 30 cycles of [98° C. for 10sec, 62° C. for 30 sec, 72° C. for 30 sec], and finally an extensioncycle at 72° C. for 5 minutes. The PCR product thus obtained was clonedinto the pT7Blue-3 Perfectly Blunt Cloning kit (Novagen, San Diego,Calif.) as per manufacturer's specifications. The recombinant plasmidswere transformed into NovaBlue competent cells (Novagen, San Diego,Calif.), and clones were sequenced. The plasmid was purified usingUltraClean 6 Minute Mini Plasmid Prep kit (MO BIO Laboratories, Inc,Solana Beach, Calif.). The plasmid thus obtained was digested withBamHI/NotI and cloned into the yeast expression vector pYES2(Invitrogen, Carlsbad, Calif.) to generate clone pYElo which was thenused for expression studies.

The Δ5-elongase full-length gene insert was 1099 bp (SEQ NO:14) inlength and, beginning with the first ATG, contained an 831 bp openreading frame encoding 276 amino acids. The amino acid sequence of thefull-length gene (SEQ ID NO:15) contained regions of homology toΔ5-elongase from Thraustochytrium sp. FJN-10, Marchantia polymorpha,Physcomitrella patens, and Mortierella alpina.

3. Example 3 Isolation of Δ4-Desaturase Nucleotide Sequences fromThraustochytrium ONC-T18

To isolate the Δ4-desaturase gene, PCR amplification was carried out ina 50 μl volume containing: 100 ng Thraustochytrium ONC-T18 genomic DNA,20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2 mM MgCl₂, 400 μM eachdeoxyribonucleotide triphosphate, 2 μM of each primer and 0.1 unit TagDNA Polymerase (Invitrogen, Carlsbad, Calif.). Thermocycling was carriedout at an annealing temperature of 59.5° C., a portion of the PCRreaction was resolved on a 0.8% agarose gel, and the band of ˜1100 bpwas purified from the remaining PCR reaction with QIAquick PCRPurification kit (Qiagen, Valencia, Calif.). This DNA fragment wascloned into the TOPO TA Cloning kit for Sequencing (Invitrogen,Carlsbad, Calif.) as per manufacturer's specifications. The recombinantplasmid was transformed into TOP10 competent cells (Invitrogen,Carlsbad, Calif.), and clones were sequenced. One clone was thusisolated that showed sequence homology to previously identifiedΔ4-desaturase. This clone is described as follows: Clone #10-3 (SEQ IDNO:16) was sequenced and the deduced amino acid sequence from 967 bpshowed 96% identity with delta-4 fatty acid desaturase Thraustochytriumsp. ATCC 21685 as the highest scoring match in a BLASTx search.

To isolate the 3′-end, genome walking was carried out using APAgene GOLDGenome Walking kit (BIO S&T Montreal, Quebec) as per manufacturer'sprotocol, using genomic DNA purified from Thraustochytrium ONC-T18, andoligonucleotides 4desat3′a (SEQ ID NO:17) (5′-TTACGCTTCCAAGGACGCGGTC-3′)and 4desat3′b (SEQ ID NO:18) (5′-ATGAACAACACGCGCAAGGAGG-3′). Botholigonucleotides were designed based on the #10-3 fragment of theputative Δ4-desaturase. The PCR fragments were resolved on 0.8% low meltagarose gel, and the bands of ˜500 to 2000 bp were gel purified. PCRproducts were eluted from agarose with water and then purified withQIAquick PCR Purification kit (Qiagen, Valencia, Calif.). These DNAfragments were cloned into the pT7Blue-3 Perfectly Blunt Cloning kit(Novagen, San Diego, Calif.) as per manufacturer's specifications. Therecombinant plasmids were transformed into NovaBlue competent cells(Novagen, San Diego, Calif.), and clones were sequenced. Clone 3′D2-92(SEQ ID NO:19) contained a 782 bp insert which was identified to containthe 3′-end of the putative Δ4-desaturase gene based on sequence homologywith known Δ4-desaturase and the presence of the ‘TGA’ stop codon.

To isolate the 5′-end, genome walking was carried out using APAgene GOLDGenome Walking kit (BIO S&T, Montreal, Quebec) as per manufacturer'sprotocol, using genomic DNA purified from Thraustochytrium sp. ONC-T18,and oligonucleotides 4desat5′a (SEQ ID NO:20)(5′-CTGGATACACGTGCCCACGAAG-3′) and 4desat5′b (SEQ ID NO:21)(5′-CACATCCAGTACAACGAGCTCCAGAA-3′). Both oligonucleotides were designedbased on the #10-3 fragment of the putative Δ4-desaturase. The PCRfragments were resolved on 0.8% low melt agarose gel, and the bands of−500 to 2000 bp were gel purified. PCR products were eluted from agarosewith water and then purified with QIAquick PCR Purification kit (Qiagen,Valencia, Calif.). These DNA fragments were cloned into the pT7Blue-3Perfectly Blunt Cloning kit (Novagen, San Diego, Calif.) as permanufacturer's specifications. The recombinant plasmids were transformedinto NovaBlue competent cells (Novagen, San Diego, Calif.), and cloneswere sequenced. Clone 5′-217 (SEQ ID NO:22) was thus obtained thatcontained a 946 bp insert that contained the putative ‘ATG’ start siteof the novel Δ4-desaturase. The deduced amino acid sequence of thisfragment, when aligned with known Δ4-desaturase showed 96% identity.

This Δ4-desaturase gene was isolated in its entirety by PCRamplification using, the Thraustochytrium sp. ONC-T18 genomic DNA as atemplate, and the following oligonucleotides: ONC-T184des380F (Forward)(SEQ ID NO:23) 5′-CGATTGAGAACCGCAAGCTTT-3′ ONC-T184DES1687R (Reverse)(SEQ ID NO:24) 5′-GCAGCACTGCTGTGCTCTGGT-3′.

PCR amplification was carried out using in a 50 μl volume: 300 ng of theThraustochytrium ONC-T18 genomic DNA, 25 mM TAPS-HCl, pH 9.3, 50 mM KCl,2 mM MgCl₂, 1 mM β-mercaptoethanol, 1.5 μl DMSO, 200 μM eachdeoxyribonucleotide triphosphate, 0.5 μM of each primer and 1 unitPhusion high-fidelity DNA polymerase (Finnzymes, Espoo, Finland).Thermocycling conditions were as follows: the template was initiallydenatured at 98° C. for 30 sec, followed by 30 cycles of [98° C. for 10sec, 61° C. for 30 sec, 72° C. for 30 sec], and finally an extensioncycle at 72° C. for 5 minutes. The PCR product thus obtained was clonedinto the pT7Blue-3 Perfectly Blunt Cloning kit (Novagen, San Diego,Calif.) as per manufacturer's specifications. The recombinant plasmidswere transformed into NovaBlue competent cells (Novagen, San Diego,Calif.), and clones were sequenced, the plasmid was purified usingUltraClean 6 Minute Mini Plasmid Prep kit (MO BIO Laboratories, Inc,Solana Beach, Calif.). The plasmid thus obtained was digested withBamHI/NotI and cloned into the yeast expression vector pYES2(Invitrogen, Carlsbad, Calif.) to generate clone pYDes which was thenused for expression studies.

The Δ4-desaturase full-length gene insert was 1757 bp (SEQ ID NO:25) inlength and, beginning with the first ATG, contained an 1509 bp openreading frame encoding 519 amino acids. The amino acid sequence of thefull-length gene (SEQ ID NO:26) contained regions of homology toΔ4-desaturase from Thraustochytrium sp. ATCC21685, Thraustochytrium sp.FJN-10, and Thraustochytrium aureum. It also contained the threeconserved ‘histidine boxes’ found in all known membrane-bounddesaturases. (Okuley, et al. (1994) The Plant Cell 6:147-158). Thesewere present at amino acid 181-185, 217-222 and 454-458. As with othermembrane-bound Δ4-desaturases, the third Histidine-box motif (HXXHH(SEQID NO:.29)) in Thraustochytrium ONC-T1 8 Δ4-desaturase was found to beQXXHH(SEQ ID NO:.30). This sequence also contained a cytochrome b5domain at the 5′-end. This cytochrome is thought to function as anelectron donor in these enzymes.

4. Example 4 Expression of Thraustochytrium ONC-T18 Δ4-Desaturase andΔ5-Elongase Genes in Yeast

Clone pYDes, which consisted of the full length Δ4-desaturase clonedinto pYES2 (Invitrogen, Carlsbad, Calif.), and clone pYElo, whichconsisted of the full-length Δ5-elongase gene in pYES2, were transformedinto competent Saccharomyces cerevisiae INVSc1. Yeast transformation wascarried out using the S. c. EasyComp Transformation kit (Invitrogen,Carlsbad, Calif.) according to conditions specified by the manufacturer.Transformants were selected for uracil auxotrophy on media lackinguracil (SC-Ura). To detect the specific desaturase activity of theseclones, transformants were grown in the presence of 500 μM specificfatty acid substrates as listed below: A) Docosapentaenoic acid(22:5n-3) (conversion to docosahexaenoic acid would indicateΔ4-desaturase activity), B) Docosatetraenoic acid (22:4n-6) (conversionto docosapentaenoic acid (22:5n-6) would indicate Δ4-desaturaseactivity), C) Dihomo-gamma-linolenic acid (20:3n-6) (conversion toarachidonic acid (20:4n-6) would indicate Δ5-desaturase activity), D)Eicosapentaenoic acid (20:5n-3) (conversion to docosapentaenoic acid(22:5n-3) would indicate Δ5-elongase activity), E) Arachidonic acid(20:4n-6) (conversion to docosatetraenoic acid (22:4n-6) would indicateΔ5-elongase activity)

The negative control strain was INVSc1 containing the unaltered pYES2vector, and these were grown simultaneously. The cultures werevigorously agitated (150 rpm) and grown for 96 hours at 20° C. in thepresence of 500 μM (final concentration) of the various substrates. Thecells were pelleted and washed in 100 mM phosphate buffer, pH 7.0, cellpellets were freeze dried. The lipids were then extracted andderivitized to fatty acid methyl esters (FAME) for gas chromatographyanalysis (GC). Transesterification and extraction was done using 100 mgfreeze dried cells, with C19:0 as internal standard, addedtransesterification reaction mix (methanol:hydrochloric acid:chloroform,10:1:1) mixed and heated at 90° C. for 2 hours, then allowed to cool atroom temperature. FAMEs were extracted by adding 1 ml water, and 2 mlhexane:chloroform (4:1), and allow organic and aqueous phases toseparate. The organic layer was extracted and treated with 0.5 g ofanhydrous sodium sulfate to remove particulates and residual water. Theorganic solvents were evaporated under a stream of argon. The FAMEs wereresuspended in iso-octane and analysed by GC-FID. The percent conversionwas calculated by dividing the product produced by the sum of (theproduct produced+the substrate added) and then multiplying by 100. Table4 represents the enzyme activity of the genes isolated based on thepercent conversion of substrate added.

TABLE 4 Δ4-desaturase enzyme activity and characterization. % conversionVector Substrate Product average stdev pYES2 C22:5 n-3 C22:6 n-3 0.070.14 x = 4 pYDes 14.04 4.01 pYES2 C22:4 n-6 C22:5 n-6 0.53 0.92 x = 3pYDes 13.76 1.31 pYES2 C20:3 n-6 C20:4 n-6 0.42 0.37 x = 3 pYDes 0.870.27

The pYDes clone that contained the Δ4-desaturase gene fromThraustochytrium ONC-T18 converted 14% of the 22:5n-3 substrate to22:6n-3, as well as 14% of the 22:4n-6 substrate to 22:5n-6. Thisconfirms that the gene encodes a Δ4-desaturase. There was no background(non-specific conversion of substrate) in this case.

5. Example 5 Manipulation of ONC-T18 Δ4-Desaturase Activity with FerricCitrate

Clone pYDes, which consisted of the full length Δ4-desaturase clonedinto pYES2 (Invitrogen, Carlsbad, Calif.), was transformed intocompetent Saccharomyces cerevisiae INVSc1. The negative control strainwas INVSc1 containing the unaltered pYES2 vector, and these were grownsimultaneously. The cultures were vigorously agitated (150 RPM) andgrown for 96 hours at 20° C. in the presence of 500 μM (finalconcentration) of DPA and 0.01% ferric citrate. The cells were thentreated as described previously to determine the percent conversion ofDPA to DHA. The percent conversion, in the presence of ferric citratewas 38.70%, in this case a 2.75 fold increase of Δ4-desaturase activity.

What is claimed is:
 1. A vector comprising a selectable marker and anucleic acid sequence, wherein the nucleic acid encodes a D5 elongasehaving at least 97% identity to SEQ ID NO:15 and wherein the elongaseactivity is retained.
 2. The vector of claim 1, wherein the D5 elongasecomprises conservative changes.
 3. The vector of claim 1, wherein theisolated nucleic acid comprises SEQ ID NO:14.
 4. An isolated cell,wherein the cell comprises the vector of claim
 1. 5. The cell of claim4, wherein the cell is a eukaryote, a prokaryote, a Thraustochytrid, ayeast, or an Escherichia coli.
 6. The cell of claim 4, wherein theelongase is in the presence of an elongase substrate.
 7. The cell ofclaim 6, wherein the substrate has a concentration of at least 100 μM,200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, or 1000μM.
 8. The cell of claim 4, wherein the cell is a plant or non-humananimal cell.